The ability of M. tuberculosis to acquire resistance to first and second line drugs is emerging as a worldwide problem that threatens to undermine TB control efforts in high burden settings. Highly successful multi-drug resistant (MDR) and extensively drug resistant (XDR) strains have been identified that are readily transmitted between hosts. These strains may result from genetic alterations that lead to a hyper-mutable state which enables rapid acquisition of diverse mechanisms of drug inactivation, from compensatory mutations that restore reproductive fitness, or from genetic lesions that lead to loss of uptake of drugs into the bacterium. This project will investigate these hypotheses using genetic and metabolomic analyses of susceptible, MDR and XDR isolates from human patients. Using well-characterized M. tuberculosis (MTB) isolates obtained from existing archives as well as prospectively collected specimens, we will develop and validate laboratory tools to experimentally measure the mutability of strains of M. tuberculosis and use these tools to assess this characteristic in clinical drug sensitive and resistant strains. We will also compare the neutral evolution rates in MDR and drug-sensitive isolates to test for hyper-mutation in these M. tuberculosis populations. Finally we will assess changes in the M. tuberculosis cell wall structure that may correlate with drug resistance and virulence, using a new liquid chromatography-mass spectrometry system for simultaneously measuring thousands of lipid species.